Electroporation devices and methods

Abstract

The electroporation device has an electroporation probe (26) terminal (27) for connecting to an electrode. The foam is injected at the treatment site to displace the blood rather than mixing with the blood, thereby increasing the contact time of the higher concentration of active agent with the tissue, thereby improving the efficacy. With the foam solution, a lower concentration of the agent can be used to obtain the same therapeutic effect as its liquid counterpart, thereby reducing the incidence of side effects associated with higher concentrations. Compared to the equivalent liquid solution, the foam solution is able to more effectively electroporate cells, especially when using bipolar pulses by mitigating the increase in tissue conductivity, which is usually observed with comparable liquid solutions. More efficient cell permeabilization will lead to better results where electroporation is delivered alone or as a tool to help absorb molecules into cells.

Description

Technical Field

[0001] The present invention relates to electroporation. Background Art

[0002] Document US 2006 / 089674 (Walters et al.) and document WO 2018 / 200800 (Bowers et al.) describe electroporation devices.

[0003] Electroporation is a medical and molecular biology technique used to apply an electric field to cells to increase the permeability of the cell membrane, thereby allowing the introduction of molecules that were previously impermeable to the cell. Electroporation has many applications and can be used where its effects on the cell membrane are both reversible and irreversible. In a typical electroporation procedure, a short, intense electrical pulse is generated to transiently permeabilize the cell membrane.

[0004] Reversible electroporation can be used to achieve passive diffusion within cells, such as chemotherapy, in which the electric field is below a certain voltage threshold and allows the cell membrane to repair after treatment. Reversible electroporation can involve allowing molecules such as drugs or genes to enter cells or molecules that are normally impermeable to the substance without inducing cell death simply by the electric field. The electric field threshold for cells varies from cell to cell. In irreversible electroporation, the electric field will be greater than this voltage threshold, which will create permanent nanopores in the cell membrane, disrupting cell homeostasis, and therefore the cell will die due to a combination of apoptosis and necrosis caused by the disruption of normal cell function.

[0005] The present invention is directed to an electroporation probe driver and apparatus and method for delivering high voltage, ultrashort electroporation pulses (preferably in the microsecond range, and in some cases down to the nanosecond range) close to theoretical ideal values, with fast turn-on and turn-off times and stable amplitude when applied to a biological load.

[0006] Another object is to avoid the need for large physical dimensions of the drive device.

[0007] Another aim is to achieve a more favorable therapeutic environment around the treated cells.

[0008] References

[0009] References: BLIbey et al., Bipolar nanosecond electric pulses are less efficient at elecropermeabilization and killing cells than monopolar pulses., Biochem.Biophys.Res.Commun., vol.443, no.2, pp.568 73, Jan.2014.10.1016 / j.bbrc.2013.12.004.

[0010] References: T, J, M,O′Connor R, D.,Cancellation effect is present in high-frequency reversible andirreversible electroporation.Bioelectrochemistry.2020Apr;132:107442.doi:10.1016 / j.bioelechem.2019.107442.Epub 2019Dec24.PubMed PMID:31923714. Summary of the Invention

[0011] Typically, electroporation therapy uses pulse lengths in the range of 50–100 μsec, either alone (irreversible electroporation) or in combination with chemotherapy (electrochemotherapy) to induce cell death. Recently, shorter microsecond (<10 μsec) high-frequency irreversible electroporation and nanosecond (<1 μsec) pulses have been developed, with an offsetting effect on the degree of cell permeabilization observed with bipolar pulses compared to monopolar pulses.

[0012] In the present invention, we provide devices and methods to obtain an improved environment around the treated cells. These improvements come from our understanding that, in the short microsecond range (<10 microseconds), the use of bipolar pulses may produce an offsetting effect (reduced cell permeabilization), and this also depends in part on the conductivity of the solution around the cells. The effectiveness of cell permeabilization (producing holes in the cell membrane) produced by short bipolar electrical pulses (<10 μs) is affected by conductivity, where lower conductivity will be able to achieve more effective electric pulse-induced cell membrane permeabilization. The conductivity around the cells is partly affected by the fluid volume in the area and the local injection solution (e.g., local anesthetic or other ion-containing solutions). The increase in conductivity around the cells to be electroporated will result in higher currents, which is harmful to treatment, resulting in worse cell permeabilization and pain in the patient. In a high conductivity environment, electroporation therapy may produce gas during treatment, resulting in pressure waves (called discharges, sparks or arcs) and mechanical tissue damage with severe acoustic characteristics. The cell lysis and ion release that occurs during electroporation can itself lead to increased tissue conductivity, resulting in high currents and potential machine failure and inadequate pulse delivery.

[0013] In the following, we describe improved electroporation devices for delivering pulses and delivering substances for modifying the environment surrounding treated cells, and we describe improved probe heads for both injecting substances and applying electroporation pulses in any desired order through the same needle inserted into tissue.

[0014] The electroporation apparatus described herein includes a plurality of electroporation probe terminals, a transformer for providing a stepped-up voltage, and a switching circuit for delivering pulses to the probe terminals for electroporation. The switching circuit preferably includes switches for connecting a high voltage and a low voltage or ground level to the probe terminals, and a controller configured to control the switches according to a control scheme to deliver pulses to the probe terminals, where the pulses are delivered to a group of at least two probe electrode terminals for spaced-apart probe electrodes.

[0015] Preferably, the pulses are bipolar for use with a pair of electrodes. Preferably, each terminal in a pair of terminals and its corresponding detector terminal are sequentially driven with a voltage amplitude and grounded out of phase.

[0016] Preferably, the controller is configured to apply a ramp-up (t r ) duration until a plateau voltage amplitude in the range of 100 V to 3000 V is maintained for a time in the range of 1 μs to 5 μs, and a ramp down from said voltage (t f ) duration.

[0017] Optionally, the amplitude is in the range of 700V to 1600V, the pulse plateau duration is in the range of 1μs to 3μs, and the on-time of the delivered pulse is preferably in the range of 100μs to 300μs. Preferably, the pulses are delivered immediately and continuously. Preferably, the bipolar frequency is in the range of 100kHz to 500kHz.

[0018] Preferably, the controller is configured to impose a plateau of near zero duration after the electroporation operation and allow a ramp-down decay in the pulse, with the decay rate preferably set by a fixed or adjustable value of a resistor of the charged capacitor connected in parallel with each other.

[0019] Optionally, the controller is configured with mapping data defining the relative physical positions of the detector terminals to be connected to the terminals, and is configured to apply the pulsed voltage to the space defined by the detector electrodes.

[0020] Preferably, the controller is configured to simultaneously drive a first set of terminals having the same potential and an opposing second set of terminals having a different potential for applying charge to a plane defined by the first and second mapping detector positions.

[0021] Optionally, the first group is driven at a high potential and the second group is grounded.

[0022] Preferably, the controller is configured to momentarily reverse the direction across the plane, i.e., apply to the first group a potential previously applied to the second group, and apply to the second group a potential previously applied to the first group, in which case the first and second groups are redefined so that the charge is applied in one direction across the space and then momentarily reversed.

[0023] Preferably, the controller is configured to drive a third group and a fourth group after reversing the direction across the plane, the third group and the fourth group also defining the plane, but the direction between the third group and the fourth group is different from the direction between the first group and the second group.

[0024] Preferably, the directions are approximately orthogonal. Preferably, the mapping data is applied to a plane defined by at least one quadrilateral bounded by the four detectors. Preferably, the mapping data is applied to a plurality of quadrilaterals.

[0025] Optionally, the mapping data defines quadrilaterals, and some detectors are used to define the sides of different and adjacent quadrilaterals.

[0026] Preferably, the switching circuit comprises a switch dedicated for each voltage level of each terminal.Preferably, the switching circuit comprises a switch dedicated for the high voltage level applied as a pulse to the terminal, and a switch dedicated for connecting the terminal to ground.

[0027] Preferably, the switching circuit includes a dedicated driver circuit for each switch. Preferably, each driver circuit is individually addressable by the controller. Optionally, each driver circuit includes an independent floating power supply. Preferably, the switches include FETs and / or IGBTs.

[0028] Preferably, the controller is configured to initially perform a probe interrogation to determine a desired drive profile from a probe memory.Preferably, the probe interrogation is performed by the controller to set optimized parameters for the probe.

[0029] Preferably, the controller is configured to measure the impedance of the bioburden by applying an AC signal to the detector electrodes over a frequency spectrum. Preferably, the frequency spectrum is in the range of 1 kHz to 100 kHz. Preferably, the impedance measurement is performed prior to electroporation actuation, and the controller is configured to automatically adjust actuation parameters based on the measured impedance, preferably to achieve a current of less than 500 mA across a pair of electrodes.

[0030] We also describe an electroporation device of any example described herein and a plurality of detector electrodes connected to the detector terminals.

[0031] In one example, the detector electrode is in a detector head comprising at least one electrode needle that is hollow and has at least one opening for flowing a substance into the tissue before, during, and / or after electroporation. Preferably, the controller is configured to also control the flow of the substance to the one or more needles in the method of driving the needles with pulses.

[0032] We also describe a method of operating such a device, comprising the steps of inserting the detector electrode into a biological load, and applying bipolar voltage pulses to the electrode and the load for electroporation.

[0033] Preferably, the controller pulses the opposing detector electrodes in a bipolar manner.

[0034] Preferably, the pulse smoothing voltage amplitude duration (Δt) is in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs. Preferably, the pulse smoothing voltage amplitude is in the range of 100V to 3000V, preferably 700V to 1600V.

[0035] Preferably, the duration of the active treatment power-on time of the pulsed detector electrode is in the range of 100 μs to 300 μs, and the bipolar pulse frequency based on two consecutive pulses in one cycle is in the range of 100 kHz to 500 kHz.

[0036] Preferably, the electrodes are driven so that the current and bioburden in the detector electrode is less than 500 mA for DNA migration into the permeabilized cells.

[0037] Preferably, there is irreversible electroporation in which a hypertonic solution is injected into the site to assist the cell ablation process, and the solution may include calcium.

[0038] Preferably, calcium ions (Ca++) are injected intratumorally at a concentration in the range of 2 mMol / L to 250 mMol / L, preferably in the range of 50 mMol / L to 150 mMol / L.

[0039] Preferably, a solution containing calcium ions (Ca++) at a concentration of 2 mMol / L to 150 mMol / L is injected for transient permeabilization by electroporation, and the pulse voltage applied for electroporation is 500 V / cm and 1500 V / cm; the pulse plateau duration is 1 μs to 3 μs, and the pulse number ranges from 1000 to 10,000.

[0040] In other preferred embodiments, calcium ions (Ca++) are injected intratumorally at a concentration in the range of 2 mMol / L to 250 mMol / L, preferably in the range of 2 mMol / L to 150 mMol / L.

[0041] In other preferred embodiments, a solution containing calcium ions (Ca++) at a concentration of 2 mMol / L to 150 mMol / L is injected for transient permeabilization by electroporation, and the pulse voltage applied for electroporation is 500 V / cm and 1500 V / cm; the pulse plateau duration is 1 μs to 3 μs, and the number of pulses ranges from 1000 to 10,000.

[0042] In one example, DNA is delivered to cells in a reversible electroporation procedure as follows:

[0043] (a) Inject DNA into the site,

[0044] (b) Insert the detector electrode,

[0045] (c) driving pulses to the electrodes to permeate the cells, the driving being bipolar pulses with a frequency in the range of 100 kHz to 500 kHz; a pulse plateau duration in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs; and a voltage amplitude in the range of 100 V to 3000 V; and

[0046] (d) Low-frequency electrode drive to draw DNA or reagents into cells during the electrophoresis phase, with low-voltage pulses less than 5 V and a duration of 10 ms to 100 ms.

[0047] In one example, a drug is injected into the site and electroporation is performed to allow the drug to migrate into the cells to cause cell ablation, such as a chemotherapeutic agent.

[0048] In one example, the controller releases the stored charge in a pulse of near zero duration followed by an exponential decay following the pulsed driving of the electrodes.

[0049] In one example, a controller measures the impedance of the biological load between at least one pair of electrodes and automatically sets the pulse voltage amplitude to avoid excessive current flow during electroporation.

[0050] We also describe a method of performing electroporation in a subject, comprising the steps of injecting a fluid into a target environment surrounding cells to be treated, and delivering a pulse to the site via an electroporation probe.

[0051] For many use examples, the liquid is preferably used in the form of a foam, having liquid and gas bubbles. Preferably, the foam is delivered to achieve a higher impedance than the liquid and therefore a lower conductivity. Preferably, the foam includes a foaming agent to assist in mixing the gas. The gas in the foam may include air and / or CO2. Preferably, the volume ratio of gas to liquid is approximately in the range of 1:2 to 1:10. Preferably, the foaming agent includes one or more of albumin and human serum albumin. Preferably, the foaming agent concentration is in the range of 5% to 80% by weight (w / w). Preferably, the foaming agent includes polidocanol or sodium tetradecyl sulfate (STS).

[0052] Preferably, the liquid comprises an active therapeutic ingredient. Preferably, the ingredient comprises one or more selected from calcium, potassium, bleomycin, cisplatin, DNA, and RNA. Preferably, the therapeutic composition comprises one or more selected from calcium ions, potassium ions, bleomycin, cisplatin, DNA, and RNA.

[0053] For some applications, the liquid preferably has a concentration of ions, such as calcium or potassium ions, in a concentration of 2 mMol to 250 mMol, or more preferably 2 mMol to 150 mMol.

[0054] Preferably, the electrical pulses have a pulse length in the range of 0.05 μs to 5 μs.

[0055] Preferably, for many treatments, the electrical pulses are bipolar.

[0056] Preferably, the electrical pulses are delivered in a sequence within the "on" energization time, each sequence being in the range of 1 μs to 1000 μs, repeated up to 1000 times, and at a frequency in the range of 1 KHz to 1000 KHz.

[0057] In some examples, the treatment is electrochemotherapy, with a pulse voltage in the range of 500 V / cm to 1500 V / cm, and / or a pulse duration in the range of 50 microseconds to 100 microseconds, and / or a pulse frequency in the range of 1 Hz to 5000 Hz, and / or a number of pulses in the range of 4 to 8.

[0058] In some embodiments, the treatment is irreversible electroporation, with a pulse voltage in the range of 1500 V / cm to 3000 V / cm, and / or a pulse duration in the range of 70 to 100 microseconds, and / or a pulse frequency in the range of 0.5 Hz to 10 Hz, and / or a number of pulses in the range of 90 to 200.

[0059] In some embodiments, the treatment is high-frequency irreversible electroporation, with a pulse voltage in the range of 2500 V / cm to 5000 V / cm, and / or a pulse duration in the range of 1 microsecond to 5 microseconds, and / or a pulse frequency in the range of 100 kHz to 500 kHz, and / or a number of pulses greater than 100.

[0060] In some examples, the treatment is electroporation and electrolysis (E2), the pulse voltage is in the range of 100 V / cm to 3000 V / cm, and / or the delivered charge is greater than 100 μF, and / or the pulse has an exponentially decaying wave.

[0061] We also describe a method wherein the treatment is DNA or RNA delivery, the pulse voltage is in the range of 1 V / cm to 1000 V / cm, and / or the pulse is a unipolar square wave.

[0062] In one aspect, a liquid, preferably in the form of a foam, disperses the local anesthetic into the tissue to be treated. In one aspect, the local anesthetic comprises lidocaine (also known as "lidocaine"). In one aspect, the local anesthetic comprises lidocaine with or without epinephrine in a ratio of 5 to 20 mg / ml. In one aspect, the local anesthetic comprises mepivacaine. In one aspect, the local anesthetic comprises mepivacaine in a ratio of 10 to 30 mg / ml.

[0063] In one aspect, a liquid having an anesthetic agent, preferably in the form of a foam, is administered in conjunction with a selected molecule, such as any one or more of calcium ions, potassium ions, bleomycin, DNA.

[0064] We also describe a foam for assisting in the permeabilization or ablation of cells during electroporation.

[0065] Preferably, the foam comprises a therapeutic component. Preferably, the component comprises one or more selected from calcium, potassium, bleomycin, cisplatin, DNA, and RNA.

[0066] Preferably, the foam has a concentration of ions. Preferably, the foam comprises calcium ions at a concentration of 2 mMol to 150 mMol.

[0067] In a preferred embodiment, the foam includes an anesthetic agent bound to a selected molecule, such as any one or more of calcium ions, potassium ions, bleomycin, DNA.

[0068] We also describe an electroporation probe head comprising at least one electrode needle that is hollow and has at least one opening for allowing the flow of substances into tissue before and / or during and / or after electroporation.

[0069] Preferably, there are a plurality of needles. The needles have a maximum inner width dimension in the range of 0.1 mm to 1.8 mm and a maximum outer width dimension in the range of 0.25 mm to 2.5 mm, and the maximum width dimension of the opening is in the range of 0.05 mm to 1.5 mm.

[0070] In one example, there are multiple needles and the spacing of the needles is in the range of 2 mm to 3 cm.

[0071] The openings may be arranged in a spiral or staggered manner along the length of the needle.The openings may have a greater distribution on the side of at least one needle facing the other needle.

[0072] We also describe an electroporation device comprising a pulse generator connected to a probe head comprising at least one electrode needle that is hollow and has at least one opening for flowing a substance into tissue before and / or during and / or after electroporation.

[0073] Preferably, the device comprises a controller adapted to control the delivery of pulses to the needle and to control the delivery of substance to the needle according to a desired method.

[0074] Preferably, the controller is configured to sequentially cause a first substance to be delivered to at least some of the needles, pulse the needles, and deliver a second substance to the needles. Preferably, the first substance has a lower electrical conductivity than the second substance. In one example, the first substance comprises foam. In one example, the first substance comprises foam and the second substance comprises foam, and the first substance foam has a greater gas concentration than the second substance foam.

[0075] We also describe a method of operating an electroporation device comprising pumping a substance through a needle to flow out through an opening and applying a pulse to the needle. The method may comprise delivering the substance through the needle before and / or after applying the pulse. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only with reference to the accompanying drawings, in which:

[0077] Figure 1 is a perspective view of an electroporation apparatus;

[0078] Figure 2 is a block diagram of the electroporation apparatus;

[0079] Figure 3 A high voltage generator with a transformer is shown;

[0080] Figure 4 The pulse switch and control block are shown in detail;

[0081] Figure 5 shows the electrical power flowing through the bioload in use;

[0082] Figure 6 is a schematic diagram illustrating the electrical aspects of the detector;

[0083] Figure 7 is a flow chart of the pulse generator algorithm implemented by the controller;

[0084] Figure 8 is a schematic spatial representation of the voltage applied by the detector in the bioburden;

[0085] Figure 9 is a schematic diagram of the electroporation pulse delivered by the generator, and the parameter values ​​of the electroporation pulse are shown in Table 1 below;

[0086] Figure 10 is the image of the sample pulse captured by the CRO;

[0087] Figure 11 is a set of graphs showing tissue impedance before and after electroporation;

[0088] Figure 12 is a set of relationship graphs of tumor volume changes in four conditions for comparison purposes;

[0089] Figure 13 is a graph of muscle contraction induced by electroporation, showing the benefits of shorter duration and higher frequency;

[0090] Figure 14 is a relationship diagram for an example of a pulse waveform diagram;

[0091] Figure 15 is a graph showing the relationship between propidium iodide uptake by electroporation;

[0092] Figure 16is a graph showing the relationship between impedance and current in tissue for an injected liquid solution compared to an injected foam solution;

[0093] Figure 17 is a graph showing the relationship between cell permeabilization rate and electric field strength in low conductivity buffer and high conductivity buffer;

[0094] Figure 18 is a graph showing the relationship between cell survival rate and electric field strength in low conductivity buffer and high conductivity buffer;

[0095] Figure 19 is a schematic diagram showing a foam injection head of an electroporation device; and

[0096] Figure 20 is a graph showing the reduction in electroporation current caused by the use of foam at different voltage drive levels. DETAILED DESCRIPTION

[0097] Reference Figure 1 and 2 The electroporation device 1 comprises a main control unit 2 connected to a user interface 3 and a detector drive circuit 4, all housed in a housing 9. The main control unit 2 and the interface 3 provide user-side functions, while the drive circuit 4 provides pulses to the detector.

[0098] The driving circuit 4 includes:

[0099] a pulse controller 5 , which is isolated from the rest of the drive unit 4 by an optical isolator 18 to prevent high voltage from being inadvertently transferred to the low voltage control electronics;

[0100] A high voltage generator 6 with a transformer provides a voltage of up to 1.5 kV to the capacitors, as described below with reference to Figure 3 described in more detail;

[0101] Pulse switch control 7, as reference Figure 4 As described in more detail, an array of seven detector electrodes 26 for delivering high frequency bipolar pulses; and

[0102] Voltage setting and maintaining circuit 8.

[0103] The touch screen interface 3 is operatively coupled to the main control unit 2 which manages the generation and pulse control of the high voltage, as well as the voltage, pulse duration, polarity and direction. This level of control is achieved through a series of control circuits in blocks 5-8.

[0104] High voltage generator 6 Figure 3. The controller 5 receives a low voltage direct current (DC) input, such as 48V. This input level sets the intensity level of the resultant pulses delivered to the detector, as described below. The controller 5 adjusts the time the primary transformer is energized to control the output voltage on the capacitor 14. Feedback from this output to the controller 5 (suitably isolated by a medical-grade optocoupler 18) enables the voltage to be accurately set and maintained. Thus, the output voltage of the capacitor 14 can vary over a wide range (typically 100V to about 1.5kV).

[0105] In this example, transformers 11 and 12 step up the input voltage to 1.5 kV, which is rectified by a conventionally constructed rectifier 13. This high voltage level (now DC) appears across balancing resistors 15 and 1000 μF capacitors 14 as the output of detector electrode 26. In this example, each of the four capacitors 14 handles voltages up to 450 V. Charging time is less than 10 seconds.

[0106] Diode 13 withstands the full output voltage plus the peak reverse voltage generated by the transformer's switches, which is typically several times the forward voltage. Additionally, a high-voltage capacitor 14 is placed in series to enable operation at the required voltage. A resistor 15 in parallel with the capacitor helps balance the voltage across the capacitor. While only two windings are shown at the transformer's output for clarity, this number could be higher. The preferred range for the values ​​of these components is 2 to 10.

[0107] The high voltage generator 6 provides this level of DC voltage to the pulse switch control 7 which in turn applies this voltage in a controlled pattern to the detector electrode 26 via the terminal 27 for optimising the electroporation treatment. This will be described in more detail below.

[0108] Figure 4 and 5 Shown in greater detail are the pulse switch control 7 and the voltage setting and maintaining circuit 8. The drive circuit 22, with its independent floating power supply 20, can be individually addressed by the controller 5 so that the IGBTs 24 and 25 deliver high voltage pulses to the detector terminals 27 where the polarity and duration of the voltage supplied to the detector is optimized.

[0109] To achieve the necessary switching of the detectors, a driver circuit 22 connects each detector electrode to either a high voltage rail HV or ground GND, and the voltage duration and polarity of the pin pairs can be set in software. To achieve the required high voltages, insulated gate bipolar transistors (IGBTs) (or FETs in other examples) are used. For higher voltages, the number of transistors can be increased. Although only seven circuits are shown, the number of outputs can be increased or decreased as needed.

[0110] As described above, the pulse switch control 7 includes seven top drive circuits and seven bottom drive circuits 22, each of which has a dedicated link to the power supply 20 (mainly the capacitor 14 of the high voltage generator 6). There are a series (seven in this example) of IGBT pairs 24 / 25, each for one detector electrode terminal 27. For example, the first IGBT pair 24 / 25 ( Figure 4 The leftmost pair (in the figure) connects the first detector electrode P1 to a high voltage rail HV or to ground GND. Simultaneously, the second IGBT pair connects the detector electrode P2 to ground or to the high voltage rail HV in the opposite direction. Therefore, current can flow through the bioburden in either direction.

[0111] At any given time, one detector electrode has either a high or low voltage (+1.5 kV or -1.5 kV), and the electrode opposite it is grounded. The fact that one electrode in a pair has a voltage 1.5 kV less than the other creates a relative negative pulse. Thus, looking at a pair of opposing electrodes, the voltage is bipolar, with the relative voltage switching from +1.5 kV to -1.5 kV.

[0112] The voltage setting and maintaining circuit 8 includes a comparator and an isolation control circuit that ensures that the output voltage is maintained at a set voltage for pulse train transmission.

[0113] refer to Figure 6 The detector has a memory 40 that stores detector type, configuration and output setting data. There is a single-line type memory I / O line to the memory 50.

[0114] refer to Figure 7 , a pulse switching control algorithm 100 is implemented by the controller 5 to generate and apply pulses. There is a start operation sequence 50, in which the detector memory 40 is read to obtain the detector type, configuration, and output setting data. In the detector drive sequence 100, the low-side driver is turned on, followed by the high-side driver. The controller waits for the required pulse width and compensation time, and then turns off the associated high-side driver. The controller waits for the turn-off time and compensation time, and turns off the low-side driver. Then, if necessary, a negative polarity pulse can be generated.

[0115] The controller manages the control pulses using a two-pin type detector contact technique to rotate the voltage source 180°, as Figure 8 As shown, the pin-type detector electrodes are labeled P1 to P7 respectively.

[0116] The controller 5 interrogates the detector to initially verify that it is approved and discovers the permitted pulse profile intended for the specific application. The detector identity is evaluated and the appropriate pulse profile and sequence are defined. When the detector is plugged in, the sense line activates the microcontroller by pulling the sense line low. The microcontroller can then communicate with the memory 40 to read the detector parameters. Several copies of the parameters are stored in the detector memory 40, with error checking to verify correct readings. The detector identity read sequence is as follows:

[0117] Sensing probes are attached;

[0118] Communicate with the memory and read the first set of parameters;

[0119] Verify error detection code;

[0120] If it has been verified, the setup is complete;

[0121] If not verified, the next parameter block is read;

[0122] Verify error detection code;

[0123] If no good parameter set is found, an error message is generated;

[0124] The detector type is indicated on the screen as a visual indication to the user.

[0125] Reference again Figure 8 , the rotation of the voltage pulses shown results in full electroporation of the affected tissue. In this scheme, the electrodes are energized in pairs and can be manipulated to align the direction of the pulses in a controlled manner. Setting electrodes P1 and P2 to positive (connected to HV via the top IGBT 24) and electrodes P7 and P3 to negative (connected to GND via the bottom IGBT 25) gives Figure 8 The electric field marked as 1 in FIG. Reversing the polarity of the electrode pair (ie, P1 and P2 are negative, and P7 and P3 are positive) results in a reversed electric field. Making electrodes P1 and P7 positive and electrodes P2 and P3 negative results in Figure 8 The electric field marked 5 in the diagram crosses the electric field marked 1. Similarly, reversing the polarity of the detectors results in a reversed electric field. And so on for the other possibilities.

[0126] Cancer cells display characteristic fractal patterns, and solid tumors are composed of millions of these cells. The geometry of solid tumors can be modeled as an ellipsoid ranging from oblate to prolate. While small tumors can be treated in a single session at a single location, tumors larger than the distance between electrodes require rotating the electrodes around the tumor surface to maximize cell permeabilization and subsequent therapeutic efficacy. The device described in this article offers usability and efficiency advantages because it rotates the electric field around the tumor surface, reducing the number of detector repositioning steps.

[0127] Furthermore, the uniformity of electric field delivery to tissues in multiple directions from a single location increases the likelihood of cell membrane perforation, thereby maximizing the therapeutic effects of chemotherapy drugs such as bleomycin and cisplatin, or in combination with other therapies such as calcium, potassium, and gene therapy, thus offering clinically significant advantages.

[0128] Furthermore, it is important to note that in order to achieve optimal therapeutic efficacy, it is necessary to deliver the pulse during the pharmacokinetic peak. This device reduces the number of times the electrode needs to be repositioned and therefore reduces the time it takes to deliver the pulse to the entire tumor surface. Figure 19 and 20 As described in more detail, furthermore, through the use of electrodes, substances such as therapeutic agents can be delivered directly through apertures along the length of the electrode, which helps to optimally ensure correct and efficient delivery of the substance.

[0129] Furthermore, milder electric field conditions can be used because these fields can be rotated around the tumor surface.

[0130] Figure 8 The scheme shown is based on at least four detector electrodes arranged in a quadrilateral, thereby defining a space in the transverse plane between them. The two electrodes defining one side of the quadrilateral are positive (e.g., P1 and P7), while the two electrodes defining the opposite side are negative (e.g., P2 and P3), and the state is instantly reversed. This scheme is then performed in the orthogonal direction (e.g., P7 and P3 are positive, P1 and P2 are negative). The same pattern is repeated for the other quadrilaterals defined by electrodes P1-P6-P5-P7 and electrodes P5-P7-P3-P4. Of course, many electrodes play different roles in different quadrilaterals.

[0131] The effect of this scheme is that the tissue between the electrodes 26 is divided into three regions bounded by quadrilaterals, and each region is treated globally with four approximately orthogonal directions of charge passing through the tissue, e.g. Figure 8 As shown by the arrows in the quadrilateral.

[0132] In general, the controller is configured to simultaneously drive a first set of electrodes (e.g., P1, P7) having the same potential (e.g., 1.5 kV) and an opposing second set of electrodes (e.g., P2, P3) having a different potential (e.g., grounded) for applying charge to a plane (e.g., orthogonal) defined by the first and second electrode positions. Since the device 1 is used to drive an external electrode set, it is programmed to drive the terminals 27 at pre-set electrode positions. This pre-setting may be referred to as mapping data, which may take the form of a series of instructions for a drive scheme or may indicate physical locations. The pre-setting may come from a query to the detector and / or from user input.

[0133] The controller advantageously reverses the direction momentarily across the plane, applying the potential previously applied to the second group to the first group and vice versa, wherein the first and second groups are redefined such that a charge is applied in one direction of the plane and then immediately reversed to apply an opposite charge. Furthermore, after applying the opposite charge, the controller drives the third and fourth groups (e.g., P1, P2 and P3, P7), which also define planes, but the plane between the third and fourth groups is different from the plane between the first and second groups, and in one example is approximately orthogonal as indicated by the arrows within the quadrilateral P1-P2-P3-P7.

[0134] Another advantageous aspect of the electroporation scheme is that a ramp-up, plateau, and ramp-down sequence is used to drive pulses to a pair of detectors. Each ramp duration is less than 0.5 μs and typically less than 0.25 μs. This is due to the short on-time of the switches used and their being driven by separate driver circuits 22, each dedicated to a separate switch and individually addressed by the controller. Therefore, if the minimum duration required for each pulse for therapeutic purposes is, for example, 2 μs, the total duration required is only 2.5 μs, after which the direction is immediately reversed. Thus, for a complete cycle of one axis through the tissue, the total duration of the two 180° directions is 5 μs. By switching directions so rapidly, every 180°, optimized treatment can be achieved while minimizing patient discomfort caused by muscle contraction. In many cases, this avoids the use of general anesthetics for treating skin cancer, for example. To improve results, pulses can also be generated orthogonally across the tissue in two directions, also with a duration of 5 μs.

[0135] refer to Figure 9 , shows the main parameters of the drive pulse. In these examples, the values ​​of these parameters are as follows.

[0136] Table 1

[0137]

[0138] In the table above, Example 1 is preferred. Examples 2 and 3 are examples of different electrode configurations, with varying depths and spacings, as listed in the table. These differences in electrode configuration result in higher ramp durations. As noted in the extreme cases, although this is generally not preferred, ramp-down values ​​approaching 1 μs are possible, and ramp durations less than 0.5 μs are preferred.

[0139] refer to Figure 10 , an image of a representative pulse of a pair of opposing electrodes taken during the experiments (1000 V, 2 μs bipolar pulses) included in Table 1. Due to the position of the electrodes and the impedance of the potato tissue, ΔU is not visible on this image. s Due to the dead time, there is a slight delay of less than 1 μs to turn off one pair of transistors before the other pair turns on (preventing a direct short circuit through the system). The circuit design minimizes this dead time. The oscilloscope shows a bipolar pulse (+1000V, -1000V) where the ramp up time and ramp down time are sharp and the electroporation pulse amplitude is maintained at the expected level for the duration of the pulse. It can be identified in this image that Figure 9 Characteristics (U, t r , t f , U / 2).

[0140] As you can see, the multi-stage transformer approach and output switch provide high voltage / high current and flexible polarity control in a compact housing. The multi-stage transformer also integrates detector detection and automatic parameter setting to ensure safe delivery of dosage for the application.

[0141] Switched mode transformers 11 / 12 operate efficiently at low voltages, but in high voltage applications they are often subject to design trade-offs. This problem is avoided in the present invention, inter alia, by:

[0142] - Use transformer drive circuits designed for low dead-time losses across a wide range of operating conditions and part-to-part variations;

[0143] - using n secondary windings connected in series to deliver a predetermined voltage Vout via a modular circuit Vmod, where Vout=Vmod*n;

[0144] - Selection of transformer core material and winding material;

[0145] - A feedback circuit is used to monitor and maintain this voltage.

[0146] As will be appreciated, the device generates safe and effective high-voltage pulses with accurate and reliable control of pulse profile, timing, duration, polarity, and direction. This is particularly advantageous in ensuring effective treatment without causing burns or requiring undesirable doses of anesthetic. Also contributing to this level of control is a sensing connection probe that automatically determines its permissible operating parameters and generator parameter configuration. Particularly advantageously, the device performs a machine-initiated interrogation of the probe to verify compatibility and select the appropriate pulse generation profile from an internal lookup table.

[0147] In other examples, the main control unit 2 can generate a research interface screen that provides access to more pulse configuration setting options available under controlled conditions. This allows researchers to change pulse parameters to the maximum value allowed by the detector to study the optimization parameters.

[0148] Device usage examples

[0149] The devices described above can be used for various treatment methods of irreversible electroporation or reversible electroporation. Some of these methods can be performed using other electroporation devices known in the art, as long as they can provide the electrical parameters shown.

[0150] As mentioned in the introductory paragraph at the beginning of this specification, electroporation is a medical and molecular biology technique used to apply an electric field to cells or molecules to increase the permeability of the cell membrane, thereby allowing chemicals, drugs, or DNA to be introduced into the cell. Electroporation has many possible medical applications and can be used both reversibly and irreversibly. In a typical electroporation procedure, a short, intense electrical pulse is generated to transiently permeabilize the cell membrane.

[0151] In reversible electroporation, which can be used, for example, in chemotherapy, the electric field is below the electric field threshold, allowing the cell membrane to repair after treatment. Reversible electroporation can allow molecules, such as drugs or genes, to enter cells or molecules that are normally impermeable to the substance or substance without inducing cell death. The electric field threshold for cells varies from cell to cell.

[0152] In irreversible electroporation (for cell ablation), the electric field is greater than a certain electric field threshold, which creates permanent nanopores in the cell membrane, disrupting cellular homeostasis and thereby forcing the cell into an irreversible cell death pathway such as via apoptosis or necrosis.

[0153] We describe a method of electroporation comprising the steps of inserting probe electrodes into a biological load and applying voltage pulses to the electrodes and load to perform electroporation, and preferably, the controller applies the pulses to the opposing probe electrodes in a bipolar manner.

[0154] Preferably, the pulse plateau voltage amplitude duration (Δt) is in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs. Preferably, the pulse plateau voltage amplitude is in the range of 100 V / cm to 3000 V / cm, preferably 500 V / cm to 2000 V / cm. Alternatively, the individual pulse lengths may be less than 1 μs and in the range of 0.03-0.99 μs, with the pulse plateau voltage amplitude in the range of 5 kV / cm to 50 kV / cm, preferably 10 kV / cm to 20 kV / cm.

[0155] Preferably, the active therapy power-on time (during which the detector electrodes are pulsed) is in the range of 100 μs to 300 μs, and the bipolar pulse frequency based on two consecutive pulses per cycle is in the range of 100 kHz to 500 kHz.

[0156] The electrodes can be driven so that the current and bioburden in the detector electrode is less than 500 mA to allow migration of DNA into the permeabilized cells.

[0157] In some examples, there is irreversible electroporation, a process in which a substance such as a liquid solution, such as a hypertonic solution, is injected into the site to assist in cell ablation, and the solution may include calcium or potassium. Preferably, calcium ions (Ca++) are injected directly into the tissue at a concentration in the range of 2 mMol / L to 250 mMol / L, and preferably in the range of 2 mMol / L to 150 mMol / L.

[0158] The composition of the substance may be selected, at least in part, to produce a desired level of tissue conductivity near and between the electrodes. For example, the substance may include deionized water to reduce conductivity. Alternatively or additionally, as described below with reference to Figures 15 to 18 More specifically, the substance may include a foam solution that preferentially increases the electrical resistance (ohms) within the tissue environment compared to a more conductive liquid solution. Consequently, the current (amperes) generated during the delivery of electroporation pulses in tissue injected with the foam solution may be reduced, thereby enabling safer and more effective treatment.

[0159] Injectable substances

[0160] A solution containing calcium ions (Ca++) at a concentration between 2 mMol / L and 150 mMol / L can be injected for transient permeabilization by electroporation, with applied pulse voltages of 800 V / cm and 2000 V / cm, a pulse plateau duration of 1 μs to 3 μs, and a pulse number ranging from 1000 to 10,000. This solution containing calcium ions is only one example. Others are provided below.

[0161] In one example, DNA is delivered to cells in a reversible electroporation procedure as follows:

[0162] (a) Inject DNA into the site,

[0163] (b) Insert the detector electrode,

[0164] (c) driving pulses to the electrodes to permeabilize the cells, the drive being, for example, bipolar pulses at a frequency in the range of 100 kHz and 500 kHz; a pulse plateau duration in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs; and a voltage amplitude in the range of 100 V / cm to 3000 V / cm; and

[0165] (d) Low-frequency electrode drive, using low-voltage pulses below 5 V to draw DNA or reagents into cells during the electrophoresis phase with pulse durations of 10 ms to 100 ms.

[0166] In an example of DNA delivery, there are:

[0167] Injecting DNA into the site, and / or

[0168] Deliver one or more unipolar 800V / cm to 1600V / cm pulses, then

[0169] Bipolar treatment at high frequency HF, for example, with a frequency in the range of 100 kHz and 500 kHz; a pulse plateau duration in the range of 1 μs to 5 μs and preferably 1 μs to 3 μs; and / or a voltage amplitude in the range of 100 V / cm to 3000 V / cm; and / or

[0170] Deliver one or more unipolar pulses of 1 V / cm to 200 V / cm.

[0171] The unipolar pulse(s) for the higher voltage of 800 V to 1600 V may have a pulse duration in the range of 50 μs to 250 μs and preferably in the range of 100 μs to 200 μs. The voltage is more preferably in the range of 1000 V / cm to 1200 V / cm.

[0172] The unipolar pulse(s) at a lower voltage of 1 V / cm to 200 V / cm may have a pulse duration of 10 ms to 10 s, preferably 20 ms to 100 ms. The voltage is preferably in the range of 100 V / cm to 150 V / cm.

[0173] The second unipolar pulse(s) are preferably delivered within 1 second of the first unipolar pulse(s).

[0174] In one example, a drug is injected into the site and electroporation is performed to allow the drug (eg, a chemotherapeutic agent) to migrate into the cells to cause cell ablation.

[0175] In one example, the controller releases the stored charge in a pulse of near zero duration followed by an exponential decay following the pulsed driving of the electrodes.

[0176] The opposing electrodes are inserted into the bioburden at intervals of 1 mm to 30 mm, more preferably 1 mm to 10 mm, and more preferably 2 mm to 8 mm. The detector electrode may be a needle electrode, but is not required.

[0177] The controller of the electroporation apparatus used was programmed to provide high frequency operating parameters within the following ranges.

[0178] The pulses to the opposing detectors or electrodes are preferably bipolar.

[0179] The frequency (bipolar frequency, the period of the positive and negative pulses) is preferably in the range of 100 kHz to 500 kHz. This is referred to as "high frequency" in this specification. This corresponds to a pulse length (stationary) of approximately 1 μs to 5 μs, but for many applications, this range is more preferably in the range of 1 μs to 3 μs, and more preferably approximately 2 μs.

[0180] The voltage amplitude is in the range of 100 V / cm to 3000 V / cm, and preferably about 800 V / cm to 2000 V / cm.The electroporation pulse may be effective in the presence of a hypertonic solution or a vesicating agent, as described in more detail below.

[0181] The duration of the active treatment, in which the detector electrode is pulsed, is preferably in the range of 0.1 ms to 1 s, more preferably 0.2 ms to 10 ms.

[0182] Within this duration, pulses are delivered in bursts (once per pulse) ranging from 100µs to 300µs. For example, if the pulse length is 2µs, there will be approximately 25 to 75 pulse cycles within the on-time.

[0183] The current in the detector is less than 1A, preferably less than 500mA. However, the current is a derived value and depends on the bioburden. For DNA migration into cells, the current is more preferably less than about 500mA, but this is less critical for other methods. In some examples, when delivering high-frequency pulses, the current can be in the range of 5A to 40A. For DNA delivery, the applied voltage is preferably less than 5V and the current is less than 1A.

[0184] The agent is delivered to the site by any well-known injection technique.

[0185] Fluid injection: hypertonic therapy for cell ablation

[0186] Liquid solutions injected locally into the target tissue facilitate irreversible electroporation and cell death and enable lower voltage pulses to be effective in the high frequency drive described above. Such solutions may, for example, include calcium ions to induce cell death.

[0187] In one example, calcium ions (Ca++) are injected intratumorally at a concentration in the range of 2 mMol / L to 150 mMol / L, and preferably at the lower end of this range due to the high frequency drive described above.

[0188] Liquid injection: DNA delivery (reversible electroporation)

[0189] For reversible electroporation there are:

[0190] (a) DNA is injected into the site.

[0191] (b) Insert the detector.

[0192] (c) Perform high-frequency treatment according to the above-mentioned high-frequency parameters to permeabilize the cells. This is called the electroporation stage.

[0193] (d) Low-frequency treatment to draw DNA into cells. This is called the electrophoresis phase. During this phase, low-frequency, low-voltage pulses below 5V are applied, with pulse durations ranging from 10ms to 100ms. This is primarily due to the negative charge of DNA, which enables efficient DNA uptake into cells.

[0194] Liquid Infusion: Drug Delivery

[0195] The drug is injected into the site and electroporated to allow the drug to passively diffuse within the cell. In one example, a drug (e.g., a chemotherapeutic agent) is selected to cause cell death (ablation). The passive diffusion of the chemotherapeutic agent is achieved by the effect of high-frequency electroporation and causes cell death. For example, the drug can be bleomycin or cisplatin.

[0196] Generally, for drug delivery, unlike DNA delivery, there is no low frequency phase ((d) above).

[0197] refer to Figures 11 to 13 , these figures show:

[0198] Effects of high-frequency and standard low-frequency electroporation pulses on potato tissue.

[0199] When combined with drugs, high-frequency electroporation compared to standard low-frequency (pulse length of approximately 100 μs) in mouse tumor models, and

[0200] Due to high-frequency electroporation, muscle contraction was reduced.

[0201] Figures 11 to 13The data clearly demonstrate effective results for drug, ion, and DNA uptake at clinically controllable voltages (<1500V).

[0202] Figure 11

[0203] Healthy biological tissues exhibit a different electrical impedance than abnormal and precancerous tissues, ie, electrical current flows more easily through abnormal and precancerous tissues, and thus the measured impedance is lower than that of healthy biological tissues.

[0204] In some examples, tissue impedance and changes in tissue impedance can be measured by the device via spectroscopy. This allows the controller to automatically modify its operation and generate user information, for example, to distinguish abnormal and precancerous tissue from healthy tissue. This is done during treatment, both before and after pulse delivery, to provide diagnostic capabilities and actionable feedback to the healthcare professional administering the treatment regarding the areas treated and those to be treated. It also allows the controller to automatically adjust voltage levels to ensure that current is not excessive, thereby automatically avoiding the adverse effects of significantly lowering impedance.

[0205] The controller measures the impedance by applying an alternating current (AC) signal to the tissue at a suitable frequency spectrum (typically 1 kHz to 100 kHz). This is done immediately before and / or immediately after the high frequency electroporation drive.

[0206] Figure 11 This figure shows the effects of high-frequency (2 μs bipolar (approximately 217 kHz)) and low-frequency (100 μs monopolar (1 Hz), 70 μs monopolar (1 Hz)) electroporation pulses on potato tissue. It also demonstrates tissue impedance measurements using a potato tissue model. A pair of probe needle electrodes were inserted 0.4 cm apart, and tissue impedance was measured before and after electroporation (experiments were performed in triplicate).

[0207] Impedance changes were monitored using the following parameters:

[0208] -2μs bipolar (about 217KHz), power-on time 200 microseconds (50 pairs), 1000V / cm, 50 transmissions

[0209] -100μs unipolar (1Hz), 1000V / cm, 8 transmissions

[0210] -70μs unipolar (1Hz), 1500V / cm, 90 transmissions

[0211] The change in tissue impedance is caused by the opening of the ion gate and the release of electrolytes into the surrounding tissue after the electroporation pulse. It provides an indication of the degree of electroporation. The largest delta change occurs in the irreversible electroporation parameters of group C (right-hand curve), which changes to 130, followed by group A (left-hand curve), which changes to 118. Standard electroporation parameters provide a net change of 72 as a control (group B) (middle curve) for drug delivery. The data show the degree of tissue electroporation achieved with bipolar high-frequency parameters, although high-frequency transmission and observed muscle contraction decrease, this situation still occurs. The data show cell permeabilization, in which the high-frequency operation of the left-hand curve shows that the device achieves good permeabilization while avoiding the risk of patient discomfort caused by high-frequency operation.

[0212] Figure 12

[0213] Figure 12 Shown are the effects of high frequency (217 KHz, 2 μs pulse plateau) versus low frequency (1 Hz) electroporation in a mouse tumor model when combined with drugs.

[0214] Preclinical testing of a mouse colorectal tumor model using the device 1 is shown. Tumors were grown to 0.1 cm in mice. 3 , followed by treatment. Four groups of 6 mice were studied:

[0215] A) Control group, no treatment,

[0216] B)ECT (Electrochemical Therapy) control,

[0217] C) ePORE containing bleomycin (Device 1), and

[0218] D) ePORE containing calcium (Device 1).

[0219] ECT control (B): Standard electroporation parameters with direct injection of bleomycin (250 IU) and using a total of 8,100 μs polarity pulses delivered at a frequency of 1 Hz with an applied field strength of 1000 V / cm.

[0220] ePORE Bleomycin (C): Pulses were delivered as bipolar pulses at a frequency of 217 kHz, with direct injection of bleomycin (250 IU).

[0221] ePORE Calcium (D): Same as ePORE Bleomycin above, but using intratumoral calcium at a concentration of 9 mg / ml and pulses delivered as bipolar pulses at a frequency of 217 KHz.

[0222] Shorter ramp times (<0.25 μs to 1000 V) enable more efficient uptake of large molecules and ions (including Ca++) directly through the cell membrane and allow for more uniform electric fields across the cell membrane at higher frequencies.

[0223] In one example, DNA uptake by cell membranes was achieved using a combination of high-frequency (>200KHz) pulses and low-current (<1Amp) pulses. A 1-2 microsecond bipolar square wave high-voltage high-frequency pulse train (50 pulses) (with a power-on time of 100-200μs) was followed within one minute by a unipolar square wave low-voltage, low-current pulse of less than 5V and <250μAmps, lasting 1-10m.

[0224] The device results in enhanced DNA endocytosis and uptake, thereby improving cell transfection and DNA expression.

[0225] In another example, cell death is achieved by delivering high-frequency electrical pulses (>200 kHz) while delivering a hypertonic solution. Such a solution can be made of sucrose, glycerol, xylose, mannitol, or fructose at a concentration between 125 and 300 mMol / L. Such a solution can also include up to 99 mMol / L of Ca2+, Mg2+, or K+.

[0226] In another example, transient permeabilization is performed by electroporation at 500-1500 V / cm in a solution containing calcium ions (Ca++) at a concentration of at least 2 mMol / L; a pulse length of 0.1 μs-3 μs; a pulse number of 1000-10,000; and a pulse frequency of 200 kHz to 500 kHz.

[0227] Figure 13

[0228] This figure shows the reduction in muscle contraction due to high-frequency electroporation from device 1. Acceleration data was calculated using porcine tissue when electroporation was delivered to colorectal tissue. Muscle contraction was observed using 1000V, 2μs bipolar pulses with a gap of less than 0.5μs between each pulse (approximately 217kHz); 1000V, 2μs bipolar pulses with a gap of 2μs between each pulse (125kHz); 1000V (1Hz), 100μs monopolar pulses; and 1500V (1Hz), 100μs monopolar pulses. The peak acceleration observed at 217kHz was 0.04g; the peak acceleration observed at 125Hz was 0.08g; the peak acceleration observed at 1000V and 100μs was 0.75g; and the peak acceleration observed at 1500V and 100μs was 0.83g.

[0229] Figure 14

[0230] As described above, the apparatus drives pulses to a pair of detector electrodes in a ramp-up, plateau, and ramp-down sequence. Due to the short on-time of the switches used and their being driven by separate drive circuits 22, each ramp lasts less than 0.5 μs or preferably less than 0.25 μs.

[0231] In one variation, the controller drives at least some of the pulses with a ramp as described above, but sets the plateau period to zero before turning off the switch to effect the pulse, thereby effecting an exponentially decaying pulse. The capacitor is charged normally, and then the pulse is initiated, allowing it to discharge freely. Figure 14 The decay rate shown is controlled by the values ​​of the balancing resistor 15 and capacitor 14. Therefore, the controller drives the electrodes to U max The pulse is then switched off, i.e., the pulse duration is set to 0. Thus, at some treatment time, the electroporation device drives a pulse with a decaying exponential shape.

[0232] This operation shows benefit after high frequency operation. It effectively discharges the capacitor.

[0233] Figure 15

[0234] Flow cytometry using propidium iodide (PI) labeling was used to determine the relationship between electroporation efficiency / cell permeabilization rate and the culture medium used as cell buffer.

[0235] We evaluated the rate of cell permeabilization induced by electroporation using ultrashort bipolar pulse lengths (2 μs) over a range of field strengths (V / cm) using standard isotonic buffer (~270 mOsm / l) and hypertonic buffer (~600 mOsm / l) containing mannitol or sucrose.

[0236] The field strength (V / cm, electrode spacing) was adjusted from 500 V / cm to 1400 V / cm in 100 V intervals, and the extent of PI uptake, and thus cell permeabilization, was determined.

[0237] To evaluate the efficiency of electroporation using ultrashort pulses delivered to cells in vitro, we used flow cytometry (FacsCalibur TM , Becton Dickinson, USA) and the fluorescent dye propidium iodide (PI), which fluoresces only after binding to DNA. Due to the size of propidium iodide, the membrane of living cells is impermeable to propidium iodide, so the fluorescence emitted after propidium iodide enters the electroporated cells and subsequently binds to DNA in the cell nucleus can be detected. FacsCalibur TMThe device is able to quantify fluorescence in individual cells, which depends on the efficacy of electroporation (ie, the number of pores opened in the cell membrane due to electroporation—allowing PI to enter the cell nucleus, thereby generating fluorescence detectable from the cell).

[0238] With CELLQUEST TM FACSCalibur software TM Flow cytometry was used to analyze cell size and fluorescence. Cell samples were obtained during the completion of the electroporation experiment. Briefly, the measured ratio of electroporated to non-electroporated cells was calculated as follows. The "Collection Criteria" was set in the "Event Count or Time" position in the Acquisition and Storage dialog box, and the acquisition time was selected. The flow rate was set and the "Voltage", "Amp Gain" and threshold were adjusted to ensure that cells could be detected. Forward scatter (FSC) and side scatter (SSC) were collected in linear mode, and FL2 (PI fluorescence) was set to logarithmic mode. Data acquisition could not begin until the sample voltage stabilized. A total of 10,000 sample points (cells) were collected for each data point. FSC versus FL2-H contour plots of the negative control (non-electroporated cells) were used to define three regions: representing intact cells, cell membrane permeabilized cells, and nuclear membrane permeabilized cells. Remaining events with low channel numbers were scored as debris.

[0239] Foam injection

[0240] The substance can be in the form of a foam, which is a liquid with very small bubbles. Foam can be used to enhance the penetration effect of electroporation and is particularly beneficial for high-frequency electroporation (greater than 100 kHz). The use of foam is described in more detail below. In this specification, the relative concentrations of liquid and gas in the foam are expressed by volume at atmospheric pressure, such as when a syringe is filled with air and liquid.

[0241] The foam may be formed by any suitable means, and may in fact be done manually by the clinician in a syringe.

[0242] A major benefit of using foam for direct injection into the target tissue to be electroporated is that, if desired for the chosen molecule, the foam can act as a carrier, and compared to liquids, foam has a more favorable effect on tissue conductivity by minimizing electrical conductivity. The air or gas component of the foam bubbles has minimal electrical conductivity relative to liquids and can provide a more favorable environment, particularly with high-frequency (>100kHz) pulses, minimizing the delivered current and helping to enhance cellular permeabilization.

[0243] The use of high frequency (>100 kHz) bipolar electrical pulses facilitates direct cell ablation or cell permeabilization for passive diffusion of molecules. In some embodiments, combination with foam has benefits on the efficacy of the procedure (relative to the use of an equivalent liquid solution).

[0244] Figure 16 The figure shows a comparison of impedance and current when using liquid and foam. Liquid has lower impedance (higher conductivity) and results in a higher current (A) being delivered. Foam, on the other hand, has higher impedance / resistance and lower conductivity, resulting in a lower current (A) being delivered. When using foam, pulse widths can be within a wider range than described above, in some cases as low as 0.05 μs, and in some cases voltages can be greater than 10 kV / cm.

[0245] In one example, Figure 17 The figure shows cell permeabilization from 0% to 100% when increasing electric fields (V / cm) are applied. Using a low-conductivity buffer allows cells to be permeabilized at lower electric field strengths compared to a high-conductivity buffer. Our understanding is that the foam creates a lower conductivity around the cells than when using the corresponding liquid solution.

[0246] Figure 18 Expected cell survival at increasing electric fields (V / cm) is shown. Low conductivity results in cell death at lower field strengths compared to high conductivity buffer.

[0247] Liquids injected without foam are rapidly diluted by the circulating blood volume. This interaction with blood reduces the effectiveness of liquid solutions because binding to plasma proteins ultimately reduces the number of active molecules. Foam, on the other hand, displaces blood rather than mixing with it, increasing the contact time of higher concentrations of active agents with tissues, resulting in greater efficacy. With foams, lower concentrations of agents can be used to achieve the same therapeutic effect as their liquid counterparts, reducing the incidence of side effects associated with higher concentrations.

[0248] Due to the presence of gas bubbles such as air, the conductivity of foams is lower than corresponding liquid solutions, resulting in lower current flow, higher cell permeability, and less pain for the patient.

[0249] In some examples, foam can be generated by mixing albumin, gas, and a liquid solution, for example, in a volume ratio of 1:4:1. Preferably, the volume ratio of gas (e.g., room air or CO2 gas) to liquid is in the range of 1:2 to 1:10.

[0250] Preferably, the foam used comprises one or more of the following:

[0251] Albumin, human serum albumin; volume concentration 10-50%, preferably 15-30%;

[0252] Polydocanol (volume concentration 0.5-5%) or sodium tetradecyl sulfate (STS) (volume concentration 0.5-5%).

[0253] STS and polidocanol are sclerosing agents, while albumin is not.

[0254] Polidocanol is also a local anesthetic.

[0255] Albumin is only a vesicant, whereas polidocanol and STS are both vesicants and sclerosants (they are irritants and directly induce cell death).

[0256] The active agent (introduced molecule) in the solution may include one or more of the following:

[0257] Calcium ions, Ca++ (2 mMol to 150 mMol); Potassium (2 mMol to 100 mMol); Bleomycin; Cisplatin; DNA; and / or RNA.

[0258] Preferably, the electroporation pulse advantageously has the following parameters:

[0259] Bipolar pulses, 0.05μs to 5μs pulse length, delivered in sequences with an “on” time of 0.1μs to 1000μs per sequence, repeated up to 1000 times at frequencies of 1kHz to 1000KHz.

[0260] Injecting foam directly into the environment surrounding cells, rather than using a pure liquid substance with the same active agent, results in an environment with poor electrical conductivity, allowing for more efficient cell permeabilization, which could help improve the efficacy of electroporation-based therapeutics.

[0261] The effectiveness of cell permeabilization (creation of pores in the cell membrane) produced by short bipolar electrical pulses (<50 μs) is affected by tissue conductivity. Higher conductivity of the fluid solution surrounding the cells will result in higher currents, which are detrimental to the treatment, leading to poor cell permeabilization and pain perception in the patient.

[0262] The increased conductivity around the cells is due in part to the fluid volume in this area and the local injection of the electroporation solution, which can include selected therapeutic molecules (calcium, potassium, bleomycin, cisplatin, etc.) and high concentrations of ions.

[0263] The use of vesicants to deliver therapeutic agents can reduce the effects of high conductivity on the efficiency of electroporation pulses in penetrating cells.

[0264] Foams made primarily of gas or air have lower conductivity than corresponding liquid solutions and therefore result in lower electrical current, higher cell permeability, and less pain for the patient.

[0265] Injection of foam into the environment to be electroporated will advantageously facilitate treatment and the degree of cell permeabilization by not increasing conductivity to the extent of comparable liquid solutions. The table below lists some preferred parameter ranges for injection foam, but these ranges are advantageously applicable to liquid injection.

[0266]

[0267] Foam can also be used to facilitate the dispersion of a local anesthetic into the tissue being treated. The local anesthetic can be 5-20 mg / ml of lidocaine, with or without epinephrine. 10 to 30 mg / ml of mepivacaine is another example of a local anesthetic that can be used. The foam and local anesthetic can be administered in conjunction with a selected molecule (e.g., calcium or potassium ions, bleomycin, DNA); or they can be provided separately.

[0268] Substance injection devices (liquid or foam only)

[0269] refer to Figure 19 , an injection device is used to deliver a substance to a target tissue through a needle electrode, which can also promote uniform distribution of the injected substance through an "aperture" or opening along its conductive length. In one example, the device 200 includes a container 201 and an array of needles 202 having a wall 203 and an aperture 204. The container 201 includes any desired type of pump, such as a peristaltic pump or a syringe. The needle gauge size is preferably in the range of 14GA to 30GA (outer diameter 0.3mm to 2.1mm; inner diameter 0.15mm to 1.8mm). More generally, it is preferred that the needle has a maximum inner width dimension in the range of 0.1mm to 1.8mm and a maximum outer width dimension in the range of 0.25mm to 2.5mm. 90, Preferably, the maximum width dimension of the opening is in the range of 0.05mm to 1.5mm, and preferably, the spacing between the needles and the nearest needle is in the range of 2mm to 3cm.

[0270] In other examples, the openings may be in the form of slots or holes of any desired shape to achieve the desired flow and distribution of the injected substance. The needle may have an insulating sleeve to cover the opening and / or be electrically insulated where no voltage is applied to the patient's tissue. The sleeve, whether insulated or not, may be removed after the substance is delivered and before the pulse is applied. The pattern of the openings is preferably as follows: Figure 19 Spiral (staggered) as shown. The number of openings may be unevenly distributed, with more towards the side of the needle facing the other needle(s) / electrode(s), to help ensure that most of the volume is distributed in the space between the electrodes. A depth indicator may be used to signal the depth of the opening relative to the tissue depth.

[0271] The probe head 200 may be used with a pulse generator as described above, or with known third party electroporation generators.

[0272] The needles may all be connected to the same pump or reservoir, or may have independent pumps or valving arrangements to allow different substances to be delivered through different needles.One or more needles may have their own solid trocar tip.

[0273] The injected substance may be a foam, but may also be a pure liquid substance, such as a non-conductive substance, for example deionized water.

[0274] Test data showed that in viable test tissue (animal liver), we evaluated the current generated during the delivery of electroporation pulses at increasing voltages. The total energized "on time" of the pulse was 6 milliseconds. When the foam was injected into the tissue before the pulse, the current generated per pulse was reduced by approximately 40%. Figure 20 It shows that, in the tests, for a given pulse voltage amplitude, the electroporation current was significantly reduced. In this relationship graph, the current is in amperes.

[0275] By using Figure 19 In a probe of this type, the electrodes deliver electrical pulses and also deliver a substance (liquid or foam only) to the tissue via the aperture 204. This enables a more uniform delivery of the substance in the environment surrounding the electrode. In addition, the probe allows for the application of a coordinated substance delivery and pulsing method that can be controlled in an optimized manner by an electronic controller that instructs the pulse generator and pumps and / or valves to deliver the substance to the needle. For example, in one programmed method, an injection is first made to provide the desired conductivity level, followed by a pulse, and then an injection is made to the perforated cells. The first substance can be a foam with a relatively high level of bubbles, and the post-pulse solution can contain a therapeutic agent, such as calcium, potassium, bleomycin or cisplatin or DNA or RNA.

[0276] Prior to the delivery of the electroporation pulse, the holes 204 deliver foam or other substances (such as a non-conductive solution, e.g., deionized water) directly into the tissue, thereby reducing the current that would otherwise be generated. The higher currents generated during the procedure can negatively impact the effectiveness of the electroporation pulse in perforating the tissue. The generation of high currents also presents technical and safety challenges for protecting the patient and the generator, such as current "arcing," tissue burning, and pulse termination before the procedure is complete.

[0277] The needle electrode 202 with the aperture 204 can be used to deliver a non-conductive solution before delivering an electrical pulse; furthermore, once the electrical pulse is delivered, the same electrode can be used to immediately administer a therapeutic solution. In practice, for example, needle electrode(s) are positioned in the target tissue, and based in part on impedance feedback, foam (or other solution) is injected directly into the target tissue through the aperture in the needle electrode(s). This is followed by the delivery of an electrical pulse through the same needle electrode, and a therapeutic substance, such as calcium or a chemotherapeutic solution, can be injected directly through the needle electrode after electroporation.

[0278] This optimizes the effectiveness of the electroporation pulse while also enabling the delivery of therapeutic substances directly into the target tissue.

[0279] exist Figure 19 In the example of , there are two electrodes, a positive and a negative electrode. In other examples, there may be a bipolar single electrode where negative and positive polarity are transmitted on the same electrode. Alternatively, there may be more than 2 electrodes in an array, such as the one described above with reference to Figure 8 described.

[0280] benefit

[0281] In general, it will be appreciated that the present invention achieves very efficient electroporation with minimal risk of patient discomfort. Some of the benefits are summarised below.

[0282] DNA absorption is improved, particularly when an initial high-frequency, high-voltage pulse (100 kHz to 500 kHz; 800 V / cm to 1600 V / cm bipolar) is followed by a low-frequency, low-voltage pulse, as described above. Particularly advantageously, due to the high-frequency nature of the high-voltage pulse, it is painless for the patient. The device delivers high-frequency pulses designed to permeabilize cell membranes and internal organelles, causing ablation alone or in combination with locally injected agents / molecules. We have described calcium absorption, with low concentrations of calcium injected in liquid or foam form ranging from 2 to 150 mMol / L, preferably near the lower end of this range. Furthermore, the high-frequency pulses generate reduced currents by utilizing the foam in the environment being treated.

[0283] As can be appreciated, the device enables the safe delivery of high voltage, ultrashort pulses (0.1 μs to 5.0 μs range) to human or animal subjects while eliminating the inefficiencies inherent in switch-mode transformer designs.

[0284] The device also enables the delivery of ultrashort pulses (microseconds) into hypertonic solutions, resulting in more efficient cell electroporation.

[0285] Furthermore, the device is able to combine ultrashort pulses (microseconds) with sharp on and off times to maximize synergistic effects, followed by capacitive discharge in the range of 10-500 μF to induce localized cell death. Particularly beneficially, the plateau is stable within a significant but brief duration of 1 μs to 3 μs, enabling very effective therapy with short on-times.

[0286] Furthermore, the device and method achieve accurate coverage of tissue with a sufficiently large electric field, avoiding the problem of uneven electric field distribution in tumor tissue due to its heterogeneity.

[0287] Lower electrical conductivity is achieved by using appropriate substances injected into the tissue, such as deionized liquids and / or foam. The lower the conductivity, the stronger the electric field. Because the distribution of the electric field in the tissue is determined by the current passing through the tissue, the above-described methods and devices help to produce a more uniform electric field, resulting in more consistent results.

[0288] The invention is not limited to the embodiments described but may be varied in construction and detail.

Claims

1. An electroporation device comprising: an electroporation probe having a plurality of terminals (27); A transformer used to provide a stepped-up voltage, a switching circuit (7) with switches (24, 25) for connecting a high voltage and a low voltage or a ground level to the plurality of terminals (27); and a controller configured to control the switch according to a control scheme to deliver a pulsed voltage to each group of at least two of the plurality of terminals (27) for electrodes spaced apart from each other, and The pulse voltage has a slope rise of less than 0.5 μs (t r ), a duration of 1 μs to 5 μs until a stable voltage amplitude in the range of 100 V to 3000 V is reached, and a ramp-down time of less than 0.5 μs (t f ) duration.

2. The electroporation device according to claim 1, wherein The pulses are bipolar for application to a pair of electrodes.

3. The electroporation device according to claim 1 or 2, wherein: Each terminal in each group is sequentially driven at a certain voltage amplitude and each terminal is grounded out of phase with its corresponding terminal.

4. The electroporation device according to claim 1 or 2, wherein: The plateau voltage amplitude is in the range of 700V to 1600V and lasts for a time in the range of 1 μs to 3 μs.

5. The electroporation device according to claim 4, wherein The on-time of the voltage of the transmission pulse is in the range of 100 μs to 300 μs.

6. The electroporation device according to claim 1 or 2, wherein: The pulsed voltage is delivered instantaneously and continuously.

7. The electroporation device according to claim 2, wherein: The bipolar frequency of the pulses is in the range of 100kHz to 500kHz.

8. The electroporation device according to claim 1 or 2, wherein: The controller is configured to allow the voltage of the pulse to ramp down with a duration close to zero after the electroporation operation.

9. The electroporation device according to claim 8, wherein The decay rate is set by fixed or adjustable values ​​of charged capacitors and resistors connected in parallel with each other.

10. The electroporation device according to claim 1 or 2, wherein: The controller is configured with mapping data defining relative physical positions of electrodes to be respectively connected to the plurality of terminals (27), and is configured to apply a pulsed voltage to a space defined by the electrodes.

11. The electroporation device according to claim 10, wherein The controller is configured to drive a first set of electrodes at a first potential and simultaneously drive an opposing second set of electrodes at a second potential for applying an electric charge to a plane defined by the first and second sets of electrodes.

12. The electroporation device according to claim 11, wherein The first set of electrodes is driven at a high potential, and the second set of electrodes is grounded.

13. The electroporation device according to claim 11, wherein The controller is configured to immediately subsequently drive the second set of electrodes at the first potential and simultaneously drive the opposing first set of electrodes at the second potential for applying opposite charges to a plane defined by the first and second sets of electrodes.

14. The electroporation device according to claim 13, wherein The controller is configured to drive the third and fourth groups of electrodes after applying the reverse charge, but a plane defined by the third and fourth groups of electrodes is different from a plane defined by the first and second groups of electrodes.

15. The electroporation device according to claim 14, wherein A plane defined by the third and fourth groups of electrodes is approximately orthogonal to a plane defined by the first and second groups of electrodes.

16. The electroporation device according to claim 10, wherein Mapping data is applied to a plane defined by at least one quadrilateral bounded by four electrodes.

17. The electroporation device according to claim 16, wherein The mapping data is applied to multiple quadrilaterals.

18. The electroporation device according to claim 17, wherein Some electrodes define sides of different and adjacent quadrilaterals.

19. The electroporation device according to claim 1 or 2, wherein: The switching circuit (7) includes switches dedicated to the voltage level applied to each terminal.

20. The electroporation device according to claim 19, wherein The switching circuit (7) includes a switch dedicated to the high voltage level applied to each terminal and a switch dedicated to grounding each terminal.

21. The electroporation device according to claim 19, wherein The switching circuit includes a drive circuit (22) dedicated to each switch.

22. The electroporation device according to claim 21, wherein Each driver circuit (22) is individually addressable by the controller.

23. The electroporation device according to claim 21, wherein Each drive circuit (22) includes an independent floating power supply.

24. The electroporation device according to claim 1 or 2, wherein: The switch includes a FET, and / or a GaN transistor, and / or a SiC transistor, and / or an IGBT (24, 25).

25. The electroporation device according to claim 1 or 2, wherein: The controller is configured to initially perform a probe interrogation to determine a desired driving profile from a probe memory (40).

26. The electroporation device according to claim 25, wherein Probe interrogation is performed by the controller to set optimized parameters for the probe.

27. The electroporation device according to claim 1 or 2, wherein: The controller is configured to measure impedance in the biological load by applying an AC signal across a frequency spectrum to the electrodes.

28. The electroporation device according to claim 27, wherein The frequency spectrum is in the range of 1 kHz to 100 kHz.

29. The electroporation device according to claim 27, wherein Impedance measurements are performed prior to electroporation actuation, and the controller is configured to automatically adjust actuation parameters based on the measured impedance.

30. The electroporation device of claim 29, wherein The driving parameters were automatically adjusted to apply less than 500 mA of current to the electrodes.

31. The electroporation device according to claim 1 or 2, wherein: The electrodes are located in a probe head (200) comprising an electrode needle (202) that is hollow and has at least one opening (204) for allowing the flow of substances into the tissue before and / or during and / or after electroporation.

32. The electroporation device of claim 31, wherein The controller is configured to control the flow (201) of the substance to the electrode needle (202) during the process of driving the electrode needle by the pulsed voltage from the electrode.

33. The electroporation device of claim 31, wherein The electrode needle (202) includes a plurality of needles.

34. The electroporation device of claim 33, wherein: The needle has a maximum inner width dimension in the range of 0.1 mm to 1.8 mm and a maximum outer width dimension in the range of 0.25 mm to 2.5 mm.

35. The electroporation device of claim 31, wherein The maximum width dimension of the opening is in the range of 0.05 mm to 1.5 mm.

36. The electroporation device of claim 31, wherein The electrode needle (202) includes a plurality of needles, and the intervals between the needles are within a range of 2 mm to 3 cm.

37. The electroporation device of claim 31, wherein: The openings are arranged in a spiral or staggered pattern along the length of the needle.

38. The electroporation device of claim 33, wherein: The openings have a greater distribution on the side of at least one needle facing the other needle.

39. The electroporation device of claim 31, wherein The controller is configured to sequentially deliver a first substance to the electrode needles, apply a pulsed voltage to the electrode needles, and deliver a second substance to the electrode needles.

40. The electroporation device of claim 39, wherein: The first substance has lower electrical conductivity than the second substance.

41. The electroporation device of claim 39, wherein the first substance comprises foam.

42. The electroporation device of claim 39, wherein: The first substance comprises foam and the second substance comprises foam, and the first substance foam has a greater gas concentration than the second substance foam.

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